Today's objective is to monitor the degradation lysozyme by our protease ( alpha-chymotrypsin) over time through measuring fluorescence of lysozyme samples incubated in the with the protease over different time intervals. This experiment is controlled through also monitoring samples containing no lysozyme in the same conditions.
Dr. Hartings's protocol for the fluorescence experiment can found in his notebook.
Stock Sample and Stock Blank Preparation
- First, a stock solution of lysozyme was made in 50µM phosphate buffer
- 5.2mg of dry lysozyme was dissolved 5mL of phosphate buffer using a 5mL volumetric flask to realize a final concentration of 1.04mg/mL (target=1.00mg/mL)
- Second, a stock solution of our protease (alpha-chymotrypsin was made)
- 1mL of HPLC grade water was added to a previously measured sample for a final stock concentration of 39.84uM
- Then, the a stock sample of an alpha-chymotrypsin and lysozyme reaction mix was made for incubation and fluorescence testing
- This sample contained 1µM protease and 974.9ul of the 1.04mg/mL lysozyme (for a total volume of 1ml)
- Through M1V1=M2V2 the volume of protease to be added from the stock solution was determined to be 25.1ul
- Volume of lysozyme to bring the final sample volume to 1mL was calculated by subtracting the volume of protease from 1ml. Thus 974.9ul of lysozyme stock was added to the 1.5mL eppendorf tube for the reaction mix.
- A made a blank solution of an alpha-chymotrypsin and phosphate buffer was then prepared to account for self degradation by the protease as well as fluorescence from the protease alone.
- Following the sample procedure, 25.1uL of protease was added to 974.9uL of phosphate buffer for a total protease concentration of 1uM.
- Both samples were then incubated in a 37˚C water bath for 1 hour.
Fluorescence Sample and Blank Standards
- 1.5mL eppendorf tubes were labeled A-H for both the blank and the lysozyme samples (16 tubes total).
- Standard A was comprised of either pure incubated blank stock or lysozyme reaction mix stock (as shown above)
- A serial dilution of both stocks for samples B-H were prepared through diluting previous standard in half with HPLC grade water and continuing this trend until standard H was diluted
- This was performed by adding 75mL of HPLC water to 75mL of the previous standard solution) and was performed with both the incubated sample stock and blank stock
- In separate 1.5 mL eppendorf tubes, fluorescence assay reaction mixes were prepared for analysis in the spectrofluorometer.
- The components of the reaction mix included
- 20µM of blank or lysozyme standard (A-H separately) 140µM of Assay Buffer and 40µM of Assay Reagent.
- With a total volume of 1mL, these fluorescence assay mixes were prepared directly before testing and added to the fluorescence cuvette Analysis with fluorometer was performed with the specifications of excitation: 390nm and emission: 400-650nm.
- Concentrations of protease in both standard solutions was as follows
Figure 1: Lysozyme Fluorescence Across a-Chymotrypsin Concentrations by Wavelength
For figure 1 below, fluorescence data for sample and blank standards A-H was corrected first by subtracted each respective blank spectra from it's sample spectra. Then the corrected absorbance at 644.5nm (the highest recorded where there should be no absorbance) was subtracted from each individual standard spectra to account for background signal. This corrected fluorescence data was plotted against the wavelength of emittance as excitation occurred at 390nm.
Figure 2: Lysozyme AuNP Fiber Digestion by alpha-Chymotrypsin Calibration Curve
For figure 2 below, the blank and background fluorescence curves were integrated to determine their area using the averaging method of summing rectangles composed of ([F1+F2]/2)*change in wavelength). In this case, F1 and F2 are adjacent fluorescence measurements within a standard spectra and the change in wavelength between readings was always .5nm. This integrated area was plotted agains the concentration of alpha-chymotrypsin in the sample standards.